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Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AβO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Glycosylation has been found to be altered in the brains of individuals with Alzheimer’s disease (AD). However, it is unknown which specific glycosylation-related pathways are altered in AD dementia. Using publicly available RNA-seq datasets covering seven brain regions and including 1724 samples, we identified glycosylation-related genes ubiquitously changed in individuals with AD. Several differentially expressed glycosyltransferases found by RNA-seq were confirmed by qPCR in a different set of human medial temporal cortex (MTC) samples (n = 20 AD vs. 20 controls). N-glycan-related changes predicted by expression changes in these glycosyltransferases were confirmed by mass spectrometry (MS)-based N-glycan analysis in the MTC (n = 9 AD vs. 6 controls). About 80% of glycosylation-related genes were differentially expressed in at least one brain region of AD participants (adjusted p-values N-glycans. Isozyme-specific changes were observed in expression of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family and the alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GALNAC) family of enzymes. Several glycolipid-specific genes (UGT8, PIGM) were upregulated. The critical transcription factors regulating the expression of N-glycosylation and elongation genes were predicted and found to include STAT1 and HSF5. The miRNA predicted to be involved in regulating N-glycosylation and elongation glycosyltransferases were has-miR-1-3p and has-miR-16-5p, respectively. Our findings provide an overview of glycosylation pathways affected by AD and potential regulators of glycosyltransferase expression that deserve further validation and suggest that glycosylation changes occurring in the brains of AD dementia individuals are highly pathway-specific and unique to AD.

High-density lipoproteins (HDL) play a critical role in cholesterol homeostasis. Apolipoprotein E (APOE), particularly the E4 allele, is a significant risk factor for Alzheimer's disease but is also a key HDL-associated protein involved in lipid transport in both the periphery and central nervous systems. The objective was to determine the influence of the APOE genotype on HDL function and size in the context of Alzheimer's disease. HDL from 194 participants (non-demented controls, mild cognitive impairment, and Alzheimer's disease dementia) were isolated from the plasma. The HDL cholesterol efflux capacity (CEC), lecithin-cholesterol acyltransferase (LCAT) activity, and particle diameter were measured. Neuropsychological test scores, clinical dementia rating, and magnetic resonance imaging scores were used to determine if cognition is associated with HDL function and size. HDL CEC and LCAT activity were reduced in APOE3E4 carriers compared to APOE3E3 carriers, regardless of diagnosis. In APOE3E3 carriers, CEC and LCAT activity were lower in patients. In APOE3E4 patients, the average particle size was lower. HDL LCAT activity and particle size were positively correlated with the neuropsychological scores and negatively correlated with the clinical dementia rating. We provide evidence for the first time of APOE genotype-specific alterations in HDL particles in Alzheimer's disease and an association between HDL function, size, and cognitive function.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These “toxic oligomers” have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AβO) and the oxidative stress elicited by AβO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer’s disease in a powerful, synergistic manner. Examination of AβO structure by various biophysical tools shows that each stilbene candidate uniquely alters AβO conformation and toxicity, providing insight towards the future development of structural correctors for AβO. Correlations of AβO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer’s, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AβO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

IntroductionThere is an increased need for the development of novel blood-based biomarkers for early detection, prevention, or intervention in Alzheimer's disease (AD). This study sought to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of AD.MethodsThe study involved 195 participants, including 96 patients with an AD diagnosis and 99 controls with no cognitive deficit. Utilizing a validated analytical mass spectrometry method, we monitored the site-specific glycosylation of 52 serum glycoproteins.ResultsPartial least-squares discriminant analysis revealed that changes in overall sialylation and fucosylation of serum glycoproteins may be indicators of an AD disease state. Loss of fucosylation of immunoglobulin G1 (IgG1) and IgG2 was indicative of AD diagnosis. Individual glycopeptide analysis found separation between the AD patients and controls on complement proteins and apolipoprotein B.DiscussionThe results of this study suggest that serum glycoprofiling may be a promising approach for biomarker discovery.

Additional file 3: Figure S2. The effect of diet and inulin on microglia bile acid signaling. Microglia were freshly isolated to extract RNA. The mRNA levels were quantified by qPCR. Data expressed as mean ± SD. n = 4 per group. *p

Additional file 2: Figure S1. Quantification of western blots showed in Figs. 2b, 3b, 4d, 5b and 7b. (A) Relative protein level of hepatic lipid biosynthesis and metabolism pathway, (B) relative protein level of hepatic inflammation, (C) relative protein level of brain homogenate, (D) relative protein level of brain inflammation, and (E) relative protein level of hepatic bile acid metabolism and synthesis. Data expressed as mean ± SD. n = 4 per group. *p

Additional file 4: Figure S3. The effect of diet and inulin on metabolomics profile. Untargeted metabolomics profile of cecal sample of CD-fed, FPC-fed, and FPC-fed with inulin supplemented mice. (A) sPLS-DA based analysis of cecal metabolomics, (B) Pathway enrichment analysis of FPC vs. CD fed and FPC + Inu fed vs. FPC fed mice based on small molecule pathway database, (C) Pathway analysis of FPC vs. CD fed and FPC + Inu. fed vs. FPC fed mice based on KEGG database, (D) Heatmap of cecal metabolites clustered with Pearson and Wald statistical analysis (n = 8 for CD-fed mice, n = 7 for FPC-fed mice, and n = 4 for inulin-supplemented FPC-fed mice).

Additional file 1: Table S1. Primers used for qPCR. Table S2. Metabolic Pathways and Function Analysis.

Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.